Staphylococcus aureus has emerged as a leading cause of life-threatening infection, both in the hospital and in the community. Beginning in 2005, deaths in the US due methicillin resistant S. aureus (MRSA) exceeded those attributable to HIV/AIDS (18,650 versus 16,000). This statistic represents only those S. aureus deaths caused by methicillin resistant strains - about half as many again were caused by penicillin resistant, methicillin sensitive strains, making the actual number of deaths in 2005 attributable to all invasive S. aureus infections over 25,000. Of invasive infections, 15% (and 8% of deaths) resulted from community acquired MRSA infection contracted with no known underlying health risk. Increasing resistance, including resistance to vancomycin, and emergence of hypervirulent strains, has heightened the importance of understanding the pathogenesis of S. aureus infection and the development of new therapeutics. We propose to use a new approach, termed TnSeq, or Tn-seq to define the key properties of S. aureus that enable it to proliferate in infection-related environments (blood, ocular fluids, abscess). TnSeq involves generating a high density transposon insertion pool (with insertions every ~35 bp around the genome). The location of transposon insertions in every cell in this pool is determined by selectively amplifying every insertion junction fragment, then sequencing these amplicons in a single Illumina reaction. The mutant pool is grown out in a variety of environmental conditions (e.g., blood, ocular fluids, or abscesses compared to laboratory medium) and the resultant output pools are resequenced and compared. This process identifies genes that, when mutated, result in a strain of S. aureus compromised in its ability to grow in one or the other ecology, and sample the genome in a massively parallel way in a single reaction. We propose to apply this technology not only for identifying genes that are essential or very important for growth in infection related environments, but also t reveal the manner in which S. aureus shifts its dependence on various metabolic pathways during the course of infection.